Z Resolution

[davek604]

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Hello All

I have a user in my facility who wants to measure the distance between 2 sub
resolution fluorescent objects in xy and z. After deconvolution we can see
there are 2 distinct objects with a small gap between them and we can
measure this gap, unfortunately it comes out roughly twice what it should be.
Would it be sensible to calibrate the error in z on our scope by imaging beads
of a definite known size and using the difference in the measured size from the
actual size as the z error. I could then subtract this error from any
measurements made.
So I have a couple of questions do beads of a known size exist as the
manufacturers I've looked at quote a size range.
Is this method even valid as Pub Med and google haven't turned up anything
similar which makes me think its probably a bad idea (or my search criteria
where rubbish).
It could also be that my measurements are correct and the objects are further
apart than first thought but I will need to prove that.
We made these measurements on a Leica SP5 using APD's emission spectrum
between 500-550nm

Any help or suggestions appreciated .

Regards

David

[mmodel]

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You may try something like in
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2010.03416.x/abstract

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Kelly
Sent: Friday, September 17, 2010 11:24 AM
To: [hidden email]
Subject: Z Resolution

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Hello All

I have a user in my facility who wants to measure the distance between 2 sub
resolution fluorescent objects in xy and z. After deconvolution we can see
there are 2 distinct objects with a small gap between them and we can
measure this gap, unfortunately it comes out roughly twice what it should be.
Would it be sensible to calibrate the error in z on our scope by imaging beads
of a definite known size and using the difference in the measured size from the
actual size as the z error. I could then subtract this error from any
measurements made.
So I have a couple of questions do beads of a known size exist as the
manufacturers I've looked at quote a size range.
Is this method even valid as Pub Med and google haven't turned up anything
similar which makes me think its probably a bad idea (or my search criteria
where rubbish).
It could also be that my measurements are correct and the objects are further
apart than first thought but I will need to prove that.
We made these measurements on a Leica SP5 using APD's emission spectrum
between 500-550nm

Any help or suggestions appreciated .

Regards

David

[Steffen Dietzel]

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David,

I am not sure I correctly understand what you attempt to do. I
understand your two objects are in the same color channel, right? So,
chromatic aberration is not an issue.

If they are sub-resolution, there is no way you can determine their size
or the position of their surface, nor can you do this for small beads.
That's the point of being sub resolution. The only thing you still can
measure accurately is the distance between their signal intensity
gravity center.

It still is a good idea to measure your point spread function with sub
resolution beads, though, since it tells you what the minimal distance
will be that you can measure = the achievable resolution. The exact size
of the beads is not so important, as long as they are well below the
resolution limit, most people I talked to use something around 170 nm.
If they get too small, they don't carry enough fluorochrome to produce
good signals.

Steffen

On 17.09.2010 17:24, David Kelly wrote:

> ============================================================
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> ============================================================
>
> Hello All
>
> I have a user in my facility who wants to measure the distance between 2 sub
> resolution fluorescent objects in xy and z. After deconvolution we can see
> there are 2 distinct objects with a small gap between them and we can
> measure this gap, unfortunately it comes out roughly twice what it should be.
> Would it be sensible to calibrate the error in z on our scope by imaging beads
> of a definite known size and using the difference in the measured size from the
> actual size as the z error. I could then subtract this error from any
> measurements made.
> So I have a couple of questions do beads of a known size exist as the
> manufacturers I've looked at quote a size range.
> Is this method even valid as Pub Med and google haven't turned up anything
> similar which makes me think its probably a bad idea (or my search criteria
> where rubbish).
> It could also be that my measurements are correct and the objects are further
> apart than first thought but I will need to prove that.
> We made these measurements on a Leica SP5 using APD's emission spectrum
> between 500-550nm
>
> Any help or suggestions appreciated .
>
> Regards
>
> David
>
>    

[Northan, Brian]

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David

Commercial Response From Media Cybernetics -

Did you have any luck calibrating the z-error in your scope that you reported a couple of weeks ago??  I work on the Autoquant deconvolution product line and it is perfectly valid to change the z spacing before a deconvolution if there is a scaling effect. The hard part is quantifying the scaling effect.

It is a mismatch between lens RI and sample RI that causes Spherical Aberration and the scaling effects in z.  What is actually happening is the PSF and the center of the PSF (z focus location) change with z.  Mike Model uses the terms "apparent" and "true" z distances.  I have also seen NFP and AFP used (Nominal and Actual Focus Positions).

Deconvolution with a z-varying psf is the true solution to the problem.  But this is computationally expensive.  The commonly used approximation (as Mike goes over in his paper) is rescaling followed by deconvolution with a stationary PSF.  And in that case you need to use the Actual or true focus locations (thus rescaling is a necessity).

In your case you report a scaling error approaching 2??  Did you confirm this??  That would be interesting.  As Mike mentions in his paper there have been conflicting results.  In the limit of low angles the scaling effect is (RI Immersion)/(RI Sample).  Some have confirmed this but other groups have reported larger scaling effects.

Brian Northan - Software Engineer, Media Cybernetics







-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Kelly
Sent: Friday, September 17, 2010 11:24 AM
To: [hidden email]
Subject: Z Resolution

============================================================
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
============================================================

Hello All

I have a user in my facility who wants to measure the distance between 2 sub
resolution fluorescent objects in xy and z. After deconvolution we can see
there are 2 distinct objects with a small gap between them and we can
measure this gap, unfortunately it comes out roughly twice what it should be.
Would it be sensible to calibrate the error in z on our scope by imaging beads
of a definite known size and using the difference in the measured size from the
actual size as the z error. I could then subtract this error from any
measurements made.
So I have a couple of questions do beads of a known size exist as the
manufacturers I've looked at quote a size range.
Is this method even valid as Pub Med and google haven't turned up anything
similar which makes me think its probably a bad idea (or my search criteria
where rubbish).
It could also be that my measurements are correct and the objects are further
apart than first thought but I will need to prove that.
We made these measurements on a Leica SP5 using APD's emission spectrum
between 500-550nm

Any help or suggestions appreciated .

Regards

David
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